The present invention relates to genes which are differentially expressed in hypertrophic cardiac tissue as compared to normal cardiac tissues. The present invention further relates to the CH-1, CH-2, CH-3, CH-4, CH-5, CH-6, CH-7, CH-8 and CH-9 genes, which genes have nucleotide homology to, but are not identical to human short-chain alcohol dehydrogenase, mouse amyloid xcex2-peptide binding protein, mouse xcex1-1 Collagen Type III, mouse xcex1-1 Collagen Type IV, chicken Collagen Type XIV, human 13kD DAP, mouse and human Gelsolin, mouse Osteonectin, mouse Transglutaminase, and mouse and human Zyxin, the proteins encoded by these genes, and derivatives and analogs thereof. This invention further provides methods of treating or preventing cardiac hypertrophy based on use of genes and the products of genes differentially expressed in hypertrophic cardiac tissue as compared to normal cardiac tissue. Additionally, the invention relates to methods of diagnosis, prognosis as well as methods of screening for modulators of the above listed proteins that are protective for cardiac hypertrophy.
Heart failure is often defined as the inability of the heart to deliver a supply of oxygenated blood sufficient to meet the metabolic needs of peripheral tissues, both at rest and during exercise. See generally, Hutter, Jr., xe2x80x9cCongestive Heart Failurexe2x80x9d, in Scientific American: Medicine, Volume 1 (1:II), eds. Dale and Federman (Scientific American, Inc. 1994). Heart failure is a common outcome of hypertension or post-myocardial infarction and is a major contributor to cardiovascular morbidity and mortality. The clinical presentation of heart failure is an energy deprived heart, with altered calcium ion homeostasis, energy metabolism, and decreased contractile reserve (see e.g, Ingwall, Circulation 87 (Suppl VII):58-62 (1993)).
Hypertrophy develops in response to chronic overloading of the heart, such as occurs in systematic hypertension or aortic stenosis. Hypertrophy entails an increase both in size of the individual muscle cells and in the overall muscle mass. While cardiac hypertrophy is thought to be an initial compensatory response to increased hemodynamic load, restoring lost function and normalizing wall stress, hypertrophy is also an independent risk factor for progression to decompensated heart failure (Kannel, in Congestive Heart Failure W. KT, Ed. (WB Saunders Co, 1989) pp. 1-9; A. M. Feldman, JAMA 267:1956-61 (1992); Katz, TCM 5:37-44 (1995); Konstam, et al., Circulation 86:431-438 (1992); Shubeita, et al., J. Biol. Chem. 265:2055-20562 (1 990)). Clinical trials in heart failure with angiotensin converting enzyme inhibitors suggest that part of the benefit of these inhibitors derives from attenuation of cardiac hypertrophy, structural remodeling and fibrosis (Chien et al., FASEB J. 5:3037-3046 (1991); Boheler et al., TCM2:176-182 (1992)).
Experiments in animal model systems and cell culture have demonstrated that cardiac hypertrophy is associated with changes in gene expression (Izumo et al., Proc. Natl. Acad. Sci. USA 85:339-343 (1988); Calderone et al., Circulation 92: 2385-2390 (1995); Boluyt, et al., Circ. Res. 75:23-32 (1994); Feldman et al., Circ. Res. 73:184-192 (1993); Buttrick et al., J. Mol. Cell. Cardiol. 26:61-67 (1994)). The specific pattern of expression differences in vivo, referred to as the molecular phenotype, is dependent on the nature of the hypertrophic stimulus, as well as the stage of compensatory hypertrophy or decompensated failure (Knowlton, et al., J. Biol. Chem. 266:7759-7768 (1991); Shubeita, et al., J. Biol. Chem. 265:20555-20562 (1990); Pennica, et al., Proc. Natl. Acad. Sci. USA 92:1142-1146 (1995); Lai, et al., Am. J. Physiol. 271:H2197-H2208 (1996); Eppenberger et al., TCM 4:187-193 (1994); Ito et al., J. Clin. Invest. 92:398-403 (1993)). Studies with cultured cardiomyocytes and non-myocytes have also suggested a role for specific mediators in the response to increased hemodynamic load including adrenergic stimulation, gp130 signaling, endothelin-1, angiotensin II, and prostaglandin F2xcex1, each with a distinct expressional and phenotypic response (Anversa et al., J. Mol. Cell. Cardiol. 12:781-795 (1980)).
Pressure overload leads to myocardial hypertrophy and the remodeling of muscular and collagenous compartments of the myocardium where the accumulation of fibrillar collagen is known to impair myocardial stiffness. Pressure overload can be achieved via surgical constriction of the aorta, aortic incompetence and aortocaval fistula (Mercadier et al., Circ. Res. 49:525-532 1981)). After abdominal aortic banding, left ventricular weight rises early and reaches a plateau by day 3 (Lindy et al., Circ. Res. 20:205-209 (1972); Turto, Cardiovasc. Res. 11:358-366 (1977)). Fibroblast proliferation also occurs with pressure overload, starting at day 2 and declining at day 7 after abdominal aortic banding (Morkin et al., Am. J. Physiol. 215:1409-1413 (1968)). Pressure overload caused cardiac hypertrophy is also associated with changes in gene expression (Lompre et al., Int. Cell Rev. 124:137-186 (1990); Schwartz et al., Heart Failure 4:154-163 (1988)). For instance, cardiac hypertrophy secondary to pressure overload is accompanied by induction of two contractile protein isogenes, B-myosin heavy chain and skeletal actin (Swynghedauw, Phydiol. Rev. 66:710-749 (1986)).
Current clinical and preclinical work suggests that cardiac hypertrophy and heart failure are problems of cardiac growth and morphogenesis. A comprehensive understanding of the molecular phenotype in compensatory and pathological hypertrophy may thus be important to developing novel therapeutic strategies as well as understanding current treatments. However, the technical limitations of the candidate gene paradigm, in which specific genes of interest have been tested one or several at a time for their association with heart disease, mean that relatively little information is available compared to the entire complement of genes expressed in the myocardium.
To obtain a more detailed understanding of the changes in gene expression occurring during pressure overload hypertrophy, the present inventors have used quantitative expression analysis (QEA) to identify expression differences in a rat surgical model of pressure overload (POL) induced cardiac hypertrophy. Abdominal aortic constriction leads to moderate hemodynamic overload (Boheler and Schwartz, TCM2:176-182 (1992); Grossman et al., J. Clin. Invest. 56:56-64 (1975)), with the heart responding by concentric hypertrophy of the left. ventricle, a process that leads to normalization of systolic wall stress (Morgan and Baker, Circulation 83:13-25 (1991)). Although increased load is thought to be the primary stimulus for this response, focal necrosis in the myocardium may also contribute to increased wall stress in this model.
This in vivo model is attractive for expression analysis as there are limited changes in tissue cellularity by infiltration of non-resident cells. The tissue reaction to acute POL is primarily due to an intrinsic response of the myocardium including hypertrophy of the cardiomyocytes, in addition to hyperplasia of endothelial, smooth muscle, and mesenchymal cells (Weber and Brilla, Circulation 83:1849-1865 (1991); Cooper, IV, Ann. Rev. Physiol. 49:501-518 (1987)). While this process is initially adaptive, there is ultimately a deterioration of contractile function accompanied by interstitial and perivascular fibrosis and increased wall stiffness (Kimura et al., Heart Circ. Physiol. 25:H1006-H1011 (1989); Batra and Rakusan, J. Cardiovasc. Pharmacol. 17 (Suppl 2):S151-S153 (1991)).
The present inventors identified the following genes that are differentially expressed in cardiac hypertrophic tissue as compared to normal cardiac tissue: genes similar to the genes encoding human short-chain alcohol dehydrogenase, mouse amyloid beta-peptide binding protein, mouse xcex1-1 Collagen Type III, mouse xcex1-1 Collagen Type IV, chicken Collagen Type XIV, human 13kD DAP, mouse and human Gelsolin, Osteonectin, mouse Transglutaminase, and mouse and human Zyxin. The present inventors also discovered that the following genes were differentially regulated in the hypertrophic tissue: xcex1-Enolase, Antizyme Inhibitor, Biglycan, Cytochrome Oxidase I, Cytochrome Oxidase II, Cyclin G, D-binding protein, Desmin, Fibrillin, Laminin, Protein kinase C-binding protein xcex215, p85, 28S rRNA gene, genes for 5.8S, 18S, and 28S RRNAS, and Preproenkephalin. The present inventors also identified the following genes which previously have been shown to be differentially expressed in hypertrophic cardiac tissue: xcex1 Skeletal Actin, ANF, ANF precursor, Atrial Natriuretic Peptide (ANP), MLC2, xcex1 Cardiac to MHC, xcex2 Cardiac MHC, and Fibronectin.
Antizyme inhibitor is a regulator of antizyme, an enzyme that accelerates the degradation of ornithine decarboxylase (ODC) by the 26 S proteasome. Antizyme inhibitor binds to the antizyme with a higher affinity than does ODC, effecting ODC release from an ODC-antizyme complex. Antizyme inhibitor contains amino acid residues required for formation of active sites of ODC, but it completely lacks ODC activity (Murakami et al., J. Biol. Chem. 271(7):3340-3342 (1996)).
Biglycan is a proteoglycan containing one or more glycosaminoglycans moieties (GAG) which are predominantly associated with the extracellular matrix (ECM). In one study, the lowest biglycan mRNA expression in mice was located within the heart, skin and kidney, while the highest was in the lungs, liver and spleen (Wegrowski et al, Genomic 30(1):8-17 (1995)). The extracellular matrix proteins collagen, fibronectin, and laminin influence biglycan mRNA expression (Dreher et al., Eur. J. Cell Biol. 53(2):296-304 (1990)).
Collagen III is distributed in skin, blood vessels, and internal organs. Chronic hypertension is known to affect collagen levels (Burgess et al., Am. J. Physiol. 270(1 Pt 2):H151-159 (1996)). Collagen III is also necessary for Collagen I fibrillogenesis in the cardiovascular region (Lui et al., Proc. Natl. Acad. Sci. USA 94(5):1852-1856 (1997)). The ratio between collagen III/I mRNA is a marker for changes in the extracellular matrix associated with hypertrophy (MRNA levels used as indicators).
Collagen XIV localizes near the surface of collagen fibrils and may be involved in epithelial-mesenchymal interactions as well as in the modulation of tissue biomechanical properties (Berthod et al., J. Invest. Dermatol. 108(5):737-742 (1997)). Collagen XIV is expressed at very few sites in the 6-day-old embryo, but occurs in virtually every collagen I-containing tissue (skeletal muscle, cardiac muscle, gizzard, tendon, periosteum, nerve) by the end of embryonic development (Walchli et al., J. Cell Sci. 107(Pt 2):669-681 (1994)).
Cyclin G contains a typical cyclin box at the N-terminus but no apparent xe2x80x9cdestruction boxxe2x80x9d or xe2x80x9cPESTxe2x80x9d sequence. Interestingly, in its C-terminus region, Cyclin G has a sequence homologous with a tyrosine phosphorylation site of the epidermal growth factor receptor. Although this cyclin is phylogenetically related to HCS26 of Saccharomyces cerevisiae, it most resembles Cig1, a B-type cyclin, of Schizosaccharomyces pombe, which has been suggested to act at the G1/S phase of the cell cycle. Cyclin G mRNA is induced within 3 hours of growth stimulation and remains elevated with no apparent cell cycle dependency (Tamura et al., Oncogene. 8(8):2113-8, (1993)).
Cytochrome C oxidase subunits I and II are two of three mitochondrial DNA encoded subunits of respiratory Complex IV (Kadenbach et al., 1983, Schoffner et al., 1995). Complex IV is located within the mitochondrial inner membrane and is the third and final enzyme of the electron transport chain of mitochondrial oxidative phosphorylation (Id.)
Desmin, an intermediate filament protein, is considered a differentiation marker for all muscle types (van Groningen et al., Biochi. Biophy. Acta 1217(1):107-109 (1994)). It is distributed throughout the cytoplasm of smooth muscle cellsxe2x80x94linking together adjacent myofibrils in heart and skeletal muscle cells. Because desmin is an intermediate filament, it can withstand larger stretching forces than actin or microtubulesxe2x80x94thereby providing tensile strength and support for the rest of the muscular system. It has been shown that contractile activity and load due to passive stretch increase desmin content in neonatal rat cardiac myocytes through increased desmin gene transcription (Watson et al., Am. J. Physiol. 270(4 Pt1):C1228-1235 ((1996)).
Fibrillin fibers are large glycoproteins that are incorporated in the outer casing of elastic fibers found within most connective tissues. Fibrillin proteins are essential for the integrity of the elastic fibers; and a mutation in the gene results in Marfan""s Syndrome (Watkins et al., Circ. Res. 60:327-336 (1987); Lockard and Bloom, J. Mol. Cell Cardiol. 25:303-309 (1993)).
Gelsolin is a calcium- and phospholipid-dependent modulator of actin. Gelsolin is also considered a marker for oligodendrocytes (Tanaka et al., Glia 19(4):286-297 (1997)), and has been shown to be active in adipose tissue (Maeda et al., Gene 190(2):227-235 (1997)).
Laminin protein is a part of the extracellular matrix, specifically an integral part of the basal lamina which endothelial cells themselves secrete (Eghbali, et al., J. Mol. Cell. Cardiol. 21:103-113 (1989)). Associated with angiogenesis, it is a fibrous protein similar to fibronectin. Laminin is related to other structural proteinsxe2x80x94collagen III (blood vessel tissue), collagen IV (basal lamina xe2x80x9cbasementxe2x80x9d membrane), and fibronectin (Engelmann et al., Mol. Cell Biochem. 163-164:47-56 (1996); Sage, Nature Med. 3:144-146 (1997)).
Secreted Protein Acidic and Rich in Cysteine (SPARC)/Osteonectin is a Ca+2 binding glycoprotein which binds to collagen. When SPARC is added to synovial fibroblasts, 3 metalloproteinase mRNAs, including collagenase, stromelysin, and gelatinase were upregulated, all of which degrade the ECM and basal laminae (Tremble et al J. Cell Biol. 121(6):1433-1444 (1993)).
The p85/ECM1 sequence has not been reported, but it has been reported to have a structural similarity to serum albumin family proteins and to Endol6 (a calcium-binding protein of sea urchin) particularly because of typical cysteine doublets (Bhalerao et al., J. Biol. Chem. 270(27):16385-94, (1995)).
Met-enkephalin and leu-enkephalin are pentapeptides which compete for and mimic the effect of opiate drugs (Legon et al., Nucleic Acids Res. 10:7905-7918 (1982)). Preproenkephalin mRNA encodes 4 copies of Met-enkephalin, 2 copies of Met-enkephalin extended sequences, and 1 copy of leu-enkephalin (Comb et al., Nature 296:663-666 (1982)). Enkephalin-deficient mice were healthy, fertile, and cared for their offsprings but displayed significant behavior abnormalities (Konig et al, Nature 383:535-538 (1996)).
Transglutaminase (TGase) is a calcium-dependent enzyme which is expressed in a variety of cells and catalyzes the cross-linking of polypeptide chains (glutamine and lysine residues in substrate proteins)xe2x80x94including ECM related proteins (Johnson et al, J. Clin. Invest. 99(12):2950-2960 (1997)). Also, chondrocyte hypertrophy results in an increase in TGase (Nurminskaya and Linsenmayer, Dev. Dyn. 206(3):260-271 (1996)). TGase binds and covalently cross-links fibronectinxe2x80x94and thereby is involved in cellular adhesion, tissue organization, and wound repair (Achyuthan et al, J. immuno. Methods 180(l):69-79 (1995)).
A component of adhesion plaques, zyxin (ZN) is involved in the microfilament organization within focal contact sites and is found primarily in cyto-skeleton associated fibroblasts. Reinhard et al, FEBS Lett. 399(1-2):103-107 (1996) found that the vasodilator-stimulated phosphoprotein (VASP) acts as a ligand for both profilin and zyxin (both involved in the adherins junctions). Although the putative designation of ZN as an attachment protein to the ECM is substantiated by numerous protein interaction studies, there are two important deviations in the amino acid structure and sequence of ZN compared to other adherin-associated proteins: 1) the amino acid sequence of ZN displays a high degree of similarity with proto-oncogenic products and transcriptional regulators (Schmeichel and Beckerle, Mol. Biol. Cell 8(2 :219-230 (1994)), and 2) there are three distinct LIM domains in ZN which are involved in metal binding (Macalma et al., J. Biol. Chem. 271(49):31470-31478 (1996)).
It should be noted that citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention.
The present inventors have applied quantitative expression analysis to a rat surgical model of cardiac hypertrophy (RCH) which produces a 60% increase in ventricle mass. The present inventors discovered that, of the 12,000 QEA-generated gene fragments derived from approximately 6,000 genes, 39 fragments (0.3%) were found to be differentially expressed in the hypertrophic hearts relative to controls. The present inventors further discovered that the expression level of a number of genes, not previously associated with cardiac hypertrophy, is significantly altered in the surgical model of cardiac hypertrophy. Expression of genes with homology to short-chain alcohol dehydrogenase, Desmin, Protein Kinase C-Binding Protein xcex215 and genes encoding 5.8S, 18S and 28S rRNAs are inhibited, while the expression of genes with homology to mouse xcex1-1 Collagen Type III, mouse xcex1-1 Collagen Type IV, chicken Collagen Type XIV, human 13kD DAP, mouse and human Gelsolin, mouse Osteonectin, mouse Transglutaminase, and mouse and human Zyxin, as well as the xcex1-Enolase, Antizyme Inhibitor, Biglycan, Cytochrome Oxidase I, Cytochrome Oxidase II, Cyclin G, Rat D-binding protein, Fibrillin, Laminin xcex3-1, p85 and Preproenkephalin genes is induced in the cardiac hypertrophy model. The above-listed genes, both the induced and inhibited genes, are collectively referred to as cardiac hypertrophy associated gene (xe2x80x9cCHAGxe2x80x9d hereinafter). Accordingly, these identified CHAG genes, RNA and proteins encoded by CHAG genes are useful in the treatment and prevention of cardiac hypertrophy, and screening for predisposition to or for protection against cardiac hypertrophy.
The present invention further relates to nucleotide sequences of CH-1, CH-2, CH-3, CH-4, CH-5, CH-6, CH-7, CH-8 and CH-9 genes, which have nucleotide sequence homology to human short-chain alcohol dehydrogenase and mouse amyloid beta-peptide binding protein, to mouse xcex1-1 Collagen Type III, to mouse xcex1-1Collagen Type IV, to chicken Collagen Type XIV, to human 13kD DAP, to mouse and human Gelsolin, to mouse Osteonectin, to mouse Transglutaminase, and mouse and human Zyxin, respectively. The present inventors have found that these genes are differentially expressed in hypertropic cardiac tissue as compared to normal cardiac tissue. Accordingly, the present invention relates to nucleotide sequences of CH-1, CH-2, CH-3, CH-4, CH-5, CH-6, CH-7, CH-8 and CH-9, and amino acid sequences of their encoded proteins, as well as derivatives (e.g., fragments) and analogs thereof. Nucleic acids hybridizable, or complementary, or in particular, inversely complementary to the foregoing nucleotide sequences are also provided. In a specific embodiment, the above-listed proteins are human proteins.
The invention also relates to derivatives (including fragments) and analogs of CH-1, CH-2, CH-3, CH-4, CH-5, CH-6, CH-7, CH-8 and CH-9 proteins which are functionally active, i.e., they are capable of displaying one or more known functional activities associated with a full-length (wild-type) protein. Such functional activities include but are not limited to antigenicity [ability to bind (or compete with the above-listed proteins for binding) to an antibody against the above-listed proteins], and immunogenicity (ability to generate antibody which binds to the above-listed proteins).
Antibodies to the above-listed proteins, and derivatives and analogs thereof, are additionally provided.
Methods of production of the proteins encoded by CH-1, CH-2, CH-3, CH-4, CH-5, CH-6, CH-7, CH-8 and CH-9, and derivatives and analogs thereof, e g., by recombinant means, are also provided.
The present invention further relates to therapeutic and diagnostic methods, particularly methods of treating, preventing, diagnosing or screening for cardiac hypertrophy, and compositions based on CHAG (CH-1, CH-2, CH-3, CH-4, CH-5, CH-6, CH-7, CH-8, CH-9, and xcex1-Enolase, Antizyme Inhibitor, Biglycan, Cytochrome Oxidase I, Cytochrome Oxidase II, Cyclin G, D-binding protein, Desmin, Fibrillin, Laminin xcex3-1, p85, Preproenkephalin, Protein Kinase C-Binding Protein xcex215, and genes encoding 5.8S, 18S and 28S rRNAs) proteins and nucleic acids. Therapeutic compounds of the invention include but are not limited to CHAG proteins and analogs and derivatives (including fragments) thereof; anti-CHAG antibodies; nucleic acids encoding the CHAG proteins, analogs, or derivatives; and CHAG antisense nucleic acids.
Animal models and methods to identify agonists and antagonists of CHAG are also provided by the invention.
CHAG: cardiac hypertrophy associated genes, identified by the present inventors, including CH-1, CH-2, CH-3, CH-4, CH-5, CH-6, CH-7, CH-8, CH-9, and xcex1-Enolase, Antizyme Inhibitor, Biglycan, Cytochrome Oxidase I, Cytochrome Oxidase II, Cyclin G, D-binding protein, Desmin, Fibrillin, Laminin xcex3-1, p85 and Preproenkephalin, Protein Kinase C-Binding Protein xcex215 and genes encoding 5.8S, 18S and 28S rRNAs.
CH: novel cardiac hypertrophy genes including: CH-1, CH-2, CH-3, CH-4, CH-5, CH-6, CH-7, CH-8 and CH-9.
QEA(copyright): Quantitative Expression Analysis.